1. Field of the Invention
This invention relates to compositions and methods for delivering camptothecin, in particular, 9-nitro-camptothecin, to patients by injection and treating diseases associated with abnormal cell proliferation such as cancer.
2. Description of Related Art
Camptothecin was isolated from the plant, Camptotheca acuminata, in the 1960""s (Wall, M. et al. (1966) J. Am. Chem. Soc. 88: 3888-3890). Camptothecin has a pentacyclic ring system with only one asymmetric center in ring E with a 20(S)-configuration. The pentacyclic ring system includes a pyrrole quinoline moiety (rings A, B and C), a conjugated pyridone (ring D), and a six-membered lactone (ring E) with an xcex1-hydroxyl group (i.e., an xcex1-hydroxy lactone).
Camptothecin itself is highly lipophilic and poorly water-soluble. Sodium camptothecin that is solubilized by sodium hydroxide in water was used in clinical trials in the early 70""s and found to have antitumor activity. However, this formulation of camptothecin administered via i.v. caused unpredictable side effects such as myelosuppression and hemorrhagic cystitis. Clinical trials with sodium camptothecin were eventually discontinued because of these toxicities and the lack of consistent antitumor activity.
Continued evaluation of this agent showed that the sodium carboxylate salt is only 10% as potent as the native camptothecin with the closed xcex1-hydroxy lactone ring intact (Wall et al. in (1969) xe2x80x9cInternational Symposium on Biochemistry and Physiology of the Alkaloids, Mothes et al. eds. Academic Verlag, Berlin, 77; Giovanella et al. (1991) Cancer Res. 51:3052). Studies also showed that camptothecin and its derivatives undergo an alkaline hydrolysis of the E-ring xcex1-hydroxy lactone, resulting in a carboxylate form of camptothecin. At pH levels below 7.0, the xcex1-hydroxy lactone E-ring form of camptothecin predominates. However, intact lactone ring E and xcex1-hydroxyl group have been shown to be essential for antitumor activity of camptothecin and its derivatives.
Camptothecin and its derivatives have been shown to inhibit DNA topoisomerase I by stabilizing the covalent complex (xe2x80x9ccleavable complexxe2x80x9d) of enzyme and strand-cleaved DNA. Inhibition of topoisomerase I by camptothecin induces protein-associated DNA single-strandbreaks which occur during the S-phase of the cell cycle. Since the S-phase is relatively short compared to other phases of the cell cycle, longer exposure to camptothecin should result in increased cytotoxicity of tumor cells. Studies indicate that only the closed xcex1-hydroxy lactone form of the drug helps stabilize the cleavable complex, leading to inhibition of the cell cycle and apoptosis.
To preserve the xcex1-hydroxy lactone form of camptothecin, camptothecin and its water insoluble derivatives have been dissolved in N-methyl-2-pyrrolidinone in the presence of an acid (U.S. Pat. No. 5,859,023). Upon dilution with an acceptable parenteral vehicle, a stable solution of camptothecin was obtained. The concentrated solution of camptothecin was also filled in gel capsules for oral administration. It is believed that such formulations increase the amount of lipophilic xcex1-hydroxy lactone form of camptothecin that diff-use through the cellular and nuclear membranes in tumor cells.
T. G. Burke, A. E. Staubus, A. K. Mishra and H. Malak (xe2x80x9cLiposomal Stabilization of Camptothecin""s Lactone Ring.xe2x80x9d J. Am. Chem. Soc. 1992, 114, 8318) and T. G. Burke, A. K. Mishra, M. C. Wani and M. E. Wall (xe2x80x9cLipid bilayer partitioning and stability of camptothecin drugs.xe2x80x9d Biochemistry 1993 May 25;32 (20):5352-64) have demonstrated that harboring of camptothecin drugs into phospholipid bilayer membranes stabilized the xcex1-hydroxy lactone moiety of camptothecin drugs against hydrolysis. In comparison to hydrolysis half-lives in PBS at 37xc2x0 C. of approximately 15 to 30 min, lipid bilayer membrane-bound camptothecin drugs were found to be stable even for periods up to 72 hours. These authors have determined an iodide ion induced quenching behavior of camptothecin""s fluorescence indicative of intercalation of camptothecin molecules between the phospholipid acyl chains of membrane bilayers, a protected environment removed from the aqueous interface. The potential for stabilization of camptothecin""s xcex1-hydroxy lactone ring structure in this environment led to the expectation that lipid bilayer intercalation might conserve the biologically active form in vivo, thereby permitting the active form to be delivered via liposomal bilayers into a biological host (U.S. Pat. No. 5,552,156).
Z Mi and T. G. Burke (xe2x80x9cDifferential interactions of camptothecin lactone and carboxylate forms with human blood components.xe2x80x9d Biochemistry 1994 Aug 30;33 (34):10325-36) exploited the intrinsic fluorescent emissions from the xcex1-hydroxy lactone and carboxylate forms of camptothecin in order to elucidate their markedly different interactions with the various components of human blood. It was found that in PBS at pH 7.4, human serum albumin (HSA) preferentially binds the carboxylate form of camptothecin with a 150-fold higher affinity than the xcex1-hydroxy lactone form. These interactions cause camptothecin""s xcex1-hydroxy lactone ring to open more rapidly and completely in the presence of HSA than in the protein""s absence. In human plasma, at pH 7.4 and 37xc2x0 C., they have observed camptothecin""s xcex1-hydroxy lactone ring to open rapidly and fully to the carboxylate form (half-life=11 min; % xcex1-hydroxy lactone at equilibrium, 0.2%). They concluded that camptothecin carboxylate""s fluorophore locates in a hydrophobic binding pocket in native HSA. In whole blood versus plasma, camptothecin""s xcex1-hydroxy lactone was found to display enhanced stability resulting in an increased half-life of 22 min and an equilibrium xcex1-hydroxy lactone concentration of 5.3%. The enhanced stability of camptothecin in human blood was found to be due to drug associations with the lipid bilayers of red blood cells. Although camptothecin xcex1-hydroxy lactone hydrolysis slows down and the equilibrium xcex1-hydroxy lactone form concentration rises on intercalation within phospholipid bilayers, the membrane-bound drug still remains thermodynamically and kinetically too labile in the presence of albumin and the concentration of the active xcex1-hydroxy lactone form in plasma remains insufficient. Thus, the liposomal bilayers cannot be considered as a pragmatic delivery system for this drug. It is speculated that a principal deactivation channel of membrane bilayer-bound camptothecin is facilitated via high affinity of the carboxylate form of the drug with albumin in-vivo compared to a relatively lower affinity of the xcex1-hydroxy lactone form of the drug with the membrane bilayers.
U.S. Pat. Nos. 5,552,156 and 5,736,156 describe liposomes and micelles of surfactant molecules for intravenous delivery of camptothecins. In liposomes, the camptothecin can reside bound to and partially in the membrane interlayer or dissociate into the internal enclosed aqueous layer in direct contact with water where the camptothecin lactone is not stable to hydrolysis. In micelles of surfactant molecules, the camptothecin is either in the central hydrocarbon portion of the micelle, bound to the micelle membrane or bound to the outside of the micelle. However, while camptothecins are less stable in micelles than in liposomes, especially in poly(ethylene oxide)-containing micelles, the amount of camptothecin compound that can bind to the membrane layer in a liposome is limited to the dimensions of the membrane and to the requirement that the membrane remain intact to prevent rupture of the liposome. The ratio of lipid to camptothecin in liposomes is generally greater than 150, and the lactone of the camptothecin slowly hydrolyzes because of the reported equilibrium between bound and free camptothecin.
The present invention provides novel injectable formulations of camptothecin, including analogs, derivatives, and pharmaceutically active metabolites of 20(S)-camptothecin, referred to herein as camptothecin compound. The present invention also provides methods of manufacturing these formulations, kits containing these formulations and methods of using these formulations to treat patients having diseases associated with abnormal cell proliferation, such as cancer.
One class of injectable formulations comprise micron and submicron size particle suspensions of a camptothecin. These micron and submicron size particle suspensions of camptothecin substantially reduce deactivation of the canptothecin by hydrolysis in-vitro and by plasma components in-vivo. The particle suspensions of the present invention display efficacy in the treatment of diseases associated with abnormal cell proliferation such as cancer when injected intravenously into mammals containing such diseases. The micron and submicron size particle suspensions of xcex1-hydroxy lactone-containing camptothecin drugs of this invention are preferably substantially stable to autoclaving, undergoing sterilization without suffering from hydrolytic deactivation of the xcex1-hydroxy lactone or substantial change in particle size. The particle suspensions can be autoclaved, cooled, and stored for long periods without undergoing agglomeration, flocculation, or aggregation.
In one embodiment, the injectable formulation comprises an aqueous suspension of solid particles suitable for intravenous delivery, the solid particles comprising a camptothecin, the solid particles having mean diameters between about 0.05 xcexcm and 10 xcexcm, the particles coated with a 0.3 xcexcm to 3.0 xcexcm thick layer of a membrane-forming amphipathic lipid.
In another embodiment, the injectable pharmaceutical composition comprises an aqueous suspension of solid particles suitable for intravenous delivery, the solid particles comprising a camptothecin, and a 0.3 nm to 3.0 xcexcm thick outer layer comprising a membrane-forming amphipathic lipid; wherein the solid particles have mean diameters between about 0.05 xcexcm and 10 xcexcm.
In yet another embodiment, an injectable pharmaceutical composition is provided which comprises: a dispersion of micrometer to submicrometer size solid particles in an aqueous carrier solution comprising one or more pharmaceutically acceptable tonicity modifier agents, the solid particles comprising a camptothecin, a first coating of not more than 10% w/w of a substantially water-insoluble, pharmaceutically acceptable lipophilic agent in which the camptothecin drug is insoluble or poorly soluble, and a second coating of at least one membrane-forming surface stabilizing amphipathic lipid, wherein the dispersion does not aggregate, flocculate, or agglomerate, and the particles do not grow in size above a volume weighted mean diameter of 10 xcexcm upon thermal sterilization.
In yet another embodiment, an injectable pharmaceutical composition is provided which comprises: a dispersion of micrometer to submicrometer size solid particles in an aqueous carrier solution comprising one or more pharmaceutically acceptable tonicity modifier agents, the solid particles comprising: a camptothecin, and a coating of at least one membrane-forming surface stabilizing amphipathic lipid, wherein the dispersion does not aggregate, flocculate, or agglomerate, and the particles do not grow in size above a volume weighted mean diameter of 10 xcexcm upon thermal sterilization.
In yet another embodiment, a pharmaceutical composition suitable for intravenous administration to a mammal is provided which comprises: an aqueous suspension of particles having mean diameters between about 0.05 xcexcm and 10 xcexcm, the particles comprising a camptothecin having an intact lactone ring, a membrane-forming amphiphatic lipid coating an outer surface of the particle, and one or more tonicity modifying agents, wherein the ratio of lipid to camptothecin in the particle is less than about 150:1 moles: mole, and wherein the mean diameters of the particles increase less than 100% upon thermal sterilization.
Another class of injectable formulations comprise an aqueous dispersion of microdroplets suitable for intravenous delivery, the microdroplets comprising a camptothecin. It is noted with regard to the microdroplet formulations that the camptothecin may be present where it is dissolved in a water-insoluble, pharmacologically acceptable liquid. The camptothecin may also be present in a solid form in addition to being dissolved in the water-insoluble, pharmacologically acceptable liquid. For example, the camptothecin may be present in the water-insoluble, pharmacologically acceptable liquid beyond the solubility of the camptothecin in the liquid.
In one embodiment, the injectable pharmaceutical composition comprises: an aqueous suspension of microdroplets suitable for intravenous delivery, the microdroplets having a mean diameter between 200 Angstroms and one micron, the microdroplets comprising a substantially water-insoluble, pharmacologically acceptable liquid, a camptothecin dissolved in the water-insoluble, pharmacologically acceptable liquid, and an outer layer comprising a phospholipid.
In another embodiment, the injectable pharmaceutical composition comprises: a dispersion in an aqueous carrier solution comprising one or more pharmaceutically acceptable tonicity modifier agents and liquid droplets of micrometer to submicrometer, the droplets comprising a substantially water-insoluble, pharmaceutically acceptable lipophilic liquid vehicle a camptothecin dissolved in the lipophilic liquid vehicle, and an outer layer surrounding the droplet comprising at least one membrane-forming amphipathic lipid, wherein upon thermal sterilization the dispersion does not aggregate, flocculate, agglomerate, or coalesce, and the droplets do not grow in size above a volume weighted mean diameter of 10 xcexcm.
In yet another embodiment, the injectable pharmaceutical composition comprises: an aqueous carrier solution comprising one or more pharmaceutically acceptable tonicity modifier agents; a dispersion of liquid droplets of a first size distribution, the liquid droplets comprising a substantially water-insoluble, pharmaceutically acceptable lipophilic liquid vehicle, solid particles of a camptothecin of a second size distribution, and an outer layer surrounding the droplet comprising at least one membrane-forming amphipathic lipid; wherein the first size distribution is in the range of submicrometer to micrometers, and the second size distribution is smaller than the first size distribution; and wherein upon thermal sterilization, the dispersion does not aggregate, flocculate, agglomerate, or coalesce, and the droplets do not grow in size above a volume weighted mean diameter of 10 xcexcm.
The camptothecins used in the formulations of the present invention may be an analog, a derivative or a pharmacologically active metabolite of 20(S)-camptothecin. In one embodiment, the camptothecin compound in the formulation has poor water solubility, for example having a water solubility of 10 xcexcg/ml or less.
Examples of the camptothecins include, but are not limited to, 20(S)-camptothecin, 9-nitro-20(S)-camptothecin, 9-amino-20(S)-camptothecin, 7-ethyl-10-(4-(1-piperdino)-1-piperdino)-carbonyloxy-camptothec in, 7-ethyl-10-hydroxy-20(S)-camptothecin, 10,11-methylenedioxy-20(S)-camptothecin, 9-chloro-20(S)-camptothecin, 9-bromo-20(S)-camptothecin, 9-hydroxy-20(S)-camptothecin, and 11-hydroxy-20(S)-camptothecin.
With regard to each of the formulations described herein, the camptothecin compound preferably contains a xcex1-hydroxy lactone ring and is most preferably 9-nitro-20(S)-camptothecin.
According to these various embodiments, the pharmaceutical composition preferably has a pH less than 7, preferably a pH less than 6 and in one embodiment, a pH between 5 and 6.
Also according to these various embodiments, the membrane-forming amphipathic lipid optionally comprises a phospholipid. The phospholipid may optionally be selected from the group consisting of saturated phospholipids, unsaturated phospholipids, synthetic phospholipids, natural phospholipids, and combinations thereof. The phospholipid may optionally be selected from the group consisting of natural and synthetic lipids, hen egg-derived phospholipid, egg phospholipid, purified egg phospholipid, soy phospholipid, dimyristoyl lecithin, didodecanoyl lecithin, dioeoyl lecithin, dilinoeoyl lecithin, alpha-palmito-beta-oleoyl lecithin, alpha-palmitoyl-beta-linoleoyl lecithin, alpha-oleoyl-beta-palmitoyl lecithin, diarachidonyl lecithin, alpha-palmito-beta-myristoyl lecithin, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, phosphatidyl serine, phosphatidyl inositol, dimyristoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol, dioctadecanoyl phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine, dihexadecyl phosphatidyl ethanolamine, dilauryl phosphatidyl ethanolamine, dimyristoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl ethanolamine, Lipoid E80, Lipoid ES, Lipoid 90H, and Lipoid 100H. In one particular variation, the phospholipid comprises Lipoid E80.
Also according to these various embodiments, the outer layer may further comprises cholesterol.
Also according to these various embodiments, the camptothecin may be present in amounts of up to about 25% w/w, more preferably up to about 5% w/w. The camptothecin may be present in amounts of from about 0.05% w/w to about 5% w/w, and optionally from about 0.1% w/w to about 1% w/w. In one particular variation, the camptothecin is present in amount of about 0.2% w/w.
Also according to these various embodiments, the membrane-forming amphipathic lipid may be present in amounts of from 0.2% w/w to about 5% w/w, more preferably in amounts of from 1% w/w to about 5% w/w. In one particular variation, the membrane-forming amphipathic lipid is present in amounts of about 4% w/w.
According to each of the above embodiments, the mean diameters of the particles and droplets preferably increase less than 100% upon storage at room temperature (23xc2x0 C.) for at least 1 month, more preferably at least 3 months, more at least 6 months, and most preferably at least 1 year.
Also according to each of the above embodiments, the lipophilic liquid vehicle is optionally present in amounts up to about 40% w/w, more preferably in amounts up to about 25% w/w, and most preferably in amounts up to about 20% w/w. The lipophilic liquid vehicle is also preferably present in amounts greater than 0.5% w/w.
The various compositions and kits of the present invention may be used in methods to treat patients suffering from abnormal cell proliferation such as cancer including hematological malignancy and tumors. In one embodiment, the method comprises: providing a pharmaceutical composition according to the present invention; and administering a therapeutically effective amount of the pharmaceutical composition intravenously to a patient in need thereof. According to the method, the camptothecin compound preferably contains a xcex1-hydroxy lactone ring and is preferably 9-nitro-20(S)-camptothecin.